Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Pat. Nos. 4,762,330, 4,723,848, 4,603,112, 5,110,587, and 5,174,993, the disclosures of which are incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome. Additional strategies have recently been reported for generating recombinant vaccinia virus.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. In the course of its history, many strains of vaccinia have arisen. These different strains demonstrate varying immunogenicity and are implicated to varying degrees with potential complications, the most serious of which are post-vaccinial encephalitis and generalized vaccinia (Behbehani, 1983).
With the eradication of smallpox, a new role for vaccinia became important, that of a genetically engineered vector for the expression of foreign genes. Genes encoding a vast number of heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990a).
The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain of vaccinia virus was protective (Morgan et al., 1988).
A fine balance between the efficacy and the safety of a vaccinia virus-based recombinant vaccine candidate is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated animal but lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology.
A number of vaccinia genes have been identified which are non-essential for growth of the virus in tissue culture and whose deletion or inactivation reduces virulence in a variety of animal systems.
The gene encoding the vaccinia virus thymidine kinase (TK) has been mapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir et al., 1983). Inactivation or complete deletion of the thymidine kinase gene does not prevent growth of vaccinia virus in a wide variety of cells in tissue culture. TK.sup.- vaccinia virus is also capable of replication in vivo at the site of inoculation in a variety of hosts by a variety of routes.
It has been shown for herpes simplex virus type 2 that intravaginal inoculation of guinea pigs with TK.sup.- virus resulted in significantly lower virus titers in the spinal cord than did inoculation with TK.sup.+ virus (Stanberry et al., 1985). It has been demonstrated that herpesvirus encoded TK activity in vitro was not important for virus growth in actively metabolizing cells, but was required for virus growth in quiescent cells (Jamieson et al., 1974).
Attenuation of TK.sup.- vaccinia has been shown in mice inoculated by the intracerebral and intraperitoneal routes (Buller et al., 1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK.sup.- recombinant vaccinia generated equivalent anti-vaccinia neutralizing antibodies as compared with the parental TK.sup.+ vaccinia virus, indicating that in this test system the loss of TK function does not significantly decrease immunogenicity of the vaccinia virus vector. Following intranasal inoculation of mice with TK.sup.- and TK.sup.+ recombinant vaccinia virus (WR strain), significantly less dissemination of virus to other locations, including the brain, has been found (Taylor et al., 1991a).
Another enzyme involved with nucleotide metabolism is ribonucleotide reductase. Loss of virally encoded ribonucleotide reductase activity in herpes simplex virus (HSV) by deletion of the gene encoding the large subunit was shown to have no effect on viral growth and DNA synthesis in dividing cells in vitro, but severely compromised the ability of the virus to grow on serum starved cells (Goldstein et al., 1988). Using a mouse model for acute HSV infection of the eye and reactivatable latent infection in the trigeminal ganglia, reduced virulence was demonstrated for HSv deleted of the large subunit of ribonucleotide reductase, compared to the virulence exhibited by wild type HSV (Jacobson et al., 1989).
Both the small (Slabaugh et al., 1988) and large (Schmidtt et al., 1988) subunits of ribonucleotide reductase have been identified in vaccinia virus. Insertional inactivation of the large subunit of ribonucleotide reductase in the WR strain of vaccinia virus leads to attenuation of the virus as measured by intracranial inoculation of mice (Child et al., 1990).
The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced (Shida, 1986). The HA gene of vaccinia virus is nonessential for growth in tissue culture (Ichihashi et al., 1971). Inactivation of the HA gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA.sup.- vaccinia virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987).
Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks on the chorioallantoic membrane of chicken eggs. Spontaneous deletions within the cowpox genome generate mutants which produce white pocks (Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDa protein encoded by an early gene (Pickup et al., 1986). This gene, which has homology to serine protease inhibitors, has been shown to inhibit the host inflammatory response to cowpox virus (Palumbo et al., 1989) and is an inhibitor of blood coagulation
The u gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Nice inoculated with a WR vaccinia virus recombinant in which the u region has been inactivated by insertion of a foreign gene produce higher antibody levels to the foreign gene product compared to mice inoculated with a similar recombinant vaccinia virus in which the u gene is intact (Zhou et al., 1990). The u region is present in a defective nonfunctional form in Copenhagen strain of vaccinia virus (open reading frames B13 and B14 by the terminology reported in Goebel et al., 1990a,b).
Cowpox virus is localized in infected cells in cytoplasmic A type inclusion bodies (ATI) (Kato et al., 1959). The function of ATI is thought to be the protection of cowpox virus virions during dissemination from animal to animal (Bergoin et al., 1971). The ATI region of the cowpox genome encodes a 160 kDa protein which forms the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987). Vaccinia virus, though containing a homologous region in its genome, generally does not produce ATI. In WR strain of vaccinia, the ATI region of the genome is translated as a 94 kDa protein (Patel et al., 1988). In Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI region are deleted, with the remaining 3' end of the region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 1990a,b).
A variety of spontaneous (Altenburger et al., 1989; Drillien et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicali et al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989; Perkus et al., 1986) deletions have been reported near the left end of the vaccinia virus genome. A WR strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al., 1988b). Specific genes within the deleted region include the virokine NIL and a 35 kDa protein (C3L, by the terminology reported in Goebel et al., 1990a,b). Insertional inactivation of NIL reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa protein is secreted like NiL into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly the complement 4B binding protein (C4 bp) (Kotwal et al., 1988a). Like the cellular C4 bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms.
The left end of the vaccinia genome includes two genes which have been identified as host range genes, K1L (Gillard et al., 1986) and C7L (Perkus et al., 1990). Deletion of both of these genes reduces the ability of vaccinia virus to grow on a variety of human cell lines (Perkus et al., 1990).
Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipox viruses. Both fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.
FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990).
Despite the host-restriction for replication of FPV and CPV to avian systems, recombinants derived from these viruses were found to express extrinsic proteins in cells of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993a,b; Taylor et al., 1992; 1991b; 1988b).
Human cytomegalovirus (HCMV) is a member of the betaherpesviridae subfamily (family Herpesviridae). HCMV is ubiquitous in humans, with usually mild or inapparent acute infection followed by persistence or latency. However, HCMV is a significant cause of morbidity and mortality in infants infected in-utero (Stagno et al., 1983). HCMV is the most common infectious complication of organ transplantation (Glenn et al., 1981) and in immunocompromised hosts (Weller et al., 1971). In AIDS patients, CMV retinitis is the leading cause of blindness (Roarty et al., 1993; Gallant et al., 1992; Gross et al., 1990) A potential role of HCMV in coronary restinosis has recently been described (Speir et al., 1994). The live attenuated Towne strain of HCMV has been shown to protect seronegative renal transplant recipients from severe clinical symptoms of HCMV infection (Plotkin et al., 1976, 1984 and 1989) and to protect initially seronegative healthy individuals from infection and clinical symptoms after subcutaneous challenge with a wild-type strain of HCMV (Plotkin et al., 1989). Concerns remain about the use of a live HCMV vaccine because of the latency reactivation phenomenon characteristic of herpesvirus infections in humans and because of the capability of certain strains of HCMV to transform cells malignantly in vitro (Albrecht and Rapp, 1973; Galloway et al., 1986). For these reasons, a recombinant subunit CMV vaccine may be more acceptable for human immunization.
The role of individual HCMV proteins in protective immunity is unclear. Three immunologically distinct families of glycoproteins associated with the HCMV envelope have been described (Gretch et al., 1988b); gCI (gp55 and gp93-130); gCII (gp47-52); and gCIII (gp85-pl45). Neutralization of HCMV has been demonstrated in vitro with antibodies specific for each of these glycoprotein families (Pachl et al., 1989; Rasmussen et al., 1988; Kari et al., 1986).
The gene coding for gCI is homologous to HSV I gB (Cranage et al., 1986). HCMVgB is synthesized as a glycosylated uncleaved precursor of apparent molecular weight 130-140 kDa which is processed by cellular proteinase into N-terminal 90-110 kDa and C-terminal 55-58 kDa products which remain associated in a disulfide linked complex (Britt and Auger, 1986; Britt and Vugler, 1989; Reis et al., 1993). Monoclonal antibodies capable of neutralizing HCMV have been obtained from mice immunized with lysates of HCMV infected cells or HCMV virions, these monoclonals were predominantly reactive with the C-terminal 55-58 kDa fragment (Britt, 1984; Kari et al., 1986; Pereira et al., 1984; Rasmussen et al., 1988). However, immunization with biochemically purified gP93 resulted in the development of gp93-specific neutralizing mAbs (Kari et al., 1990).
HCMV-gB may serve to elicit protective immunity in humans: immunization with the purified gB protein induces neutralizing antibody (Gonczol et al., 1990) and human anti-gB monoclonal antibodies neutralize the virus (Masuho et al., 1987). Following natural infection neutralizing antibody specific for HCMV-gB is observed. When gB specific antibody is absorbed from human sera, HCMV neutralizing antibody titer is reduced significantly (50-88%, Gonczol al., 1991; 0-98% median 48%, Marshall et al., 1992). There is also evidence for activation of helper T cells by the gB protein in naturally seropositive humans (Liu et al., 1991) and gB specific CTL has been detected in humans in some studies (Borysiewicz et al., 1988; Liu et al., 1991; Riddell, et al., 1991).
The gCII glycoproteins are encoded by a gene or genes in the US6 gene family (US6 through US11, Gretch et al., 1988a). These glycoproteins are recognized by human anti-HCMV antibody in sera from convalescent adults. However, sera from congenitally infected infants with persistent infection failed to react with gCII glycoproteins (Kari and Gehrz, 1990), suggesting that gCII may be important to human protective immune responses to HCMV.
The gP86 component of the gCIII complex is encoded by a gene that is homologous to HSV-I gH (Cranage et al., 1988; Pachl et al., 1989). The HCMV gH protein is capable of inducing a neutralizing immune response in humans (10% of HCMV infected individuals have a detectable level of circulating gH specific antibody (Rasmussen et al., 1991) as well as in laboratory animals (Baboonian et al., 1989; Cranage et al., 1988; Ehrlich et al., 1988; Rasmussen et al., 1984). Murine gH-specific monoclonal antibodies neutralize virus infectivity in a complement-independent manner (Baboonian et al., 1989; Cranage et al., 1988; Rasmussen et al., 1984) and inhibit viral spread (Pachl et al., 1989) suggesting that gH may be responsible for virus attachment, penetration and or spread.
Although gH is found on the surface of HCMV infected cells (Cranage et al., 1988), when expressed by a variety of recombinant systems it is restricted to the endoplasmic reticulum (Spaete et al., 1991). Coexpression of the HCMV UL115 gene product (glycoprotein gL) results in the formation of a stable complex of these two proteins and the transport of gH to the cell surface (Spaete et al., 1993; Kaye et al., 1992).
HCMV synthesizes a number of matrix tegument phosphoproteins. The pp150 phosphoprotein is highly immunogenic apparently more so than any other of the HCMV structural proteins (Jahn et al., 1987). A second matrix phosphoprotein, pp65, elicits a variable humoral response in humans (Jahn et al., 1987; Plachter et al., 1990). This protein can stimulate lymphoproliferation, IL-2 and interferon production, B-cell stimulation of antibody and natural killer cell activity (Forman et al., 1985). It also serves as a target antigen for HCMV-specific, HLA-restricted cytotoxic T cells (CTLs) (Pande et al., 1991; Gilbert et al., 1993).
Additional structural proteins may be required for eliciting a protective immune response to HCMV. The major capsid protein (UL86) is known to induce specific antibodies during natural infection and has been considered as the CMV-group common antigen (Spaete et al., 1994). The tegument phosphoprotein, pp28 (UL99), is also known to elicit persistent antibody responses during a natural infection. Further, this protein has also been implicated as a CTL target immunogen (Charpentier et al., 1986). The immune response to the upper tegument phosphoprotein, pp71 (UL82), is not as well characterized as the other tegument phosphoproteins (pp28, pp65), but as a known tegument protein requires further attention.
In addition to these structural proteins, some non-structural proteins may also be candidates for inclusion in a recombinant subunit vaccine. Immunization of mice with a recombinant vaccinia virus expressing murine cytomegalovirus (MCMV) pp89 (functional homolog of HCMV IE 1) induces CD8.sup.+ T-cell responses that mediate protective immunity from challenge with MCMV (Jonjic et al., 1988). The human CMV major immediate early protein (IE 1) has been shown to be a target for CTLs isolated from HCMV seropositive individuals (Borysiewicz et al., 1988). Since IE 1 is among the initial viral proteins expressed and is necessary for inducing the expression of other CMV genes and initiating the viral life cycle in latently infected cells (Blanton and Tevethia, 1981; Cameron and Preston, 1981; DeMarchi et al., 1980: McDonough and Spector, 1983; Wathen et al., 1981), CTL responses directed against IE 1 may be important for controlling and/or eliminating HCMV infection. Recently Gilbert et al., (1993) have suggested that HCMV has evolved a mechanism by which other viral encoded proteins selectively interfere with the presentation of IE-derived peptides in association with Class I major histocompatibility complex (MHC) molecules.
Some additional nonstructural proteins may also be candidates for inclusion in a recombinant subunit HCMV vaccine candidate. The immediate early protein, IE2 (UL122), and the regulatory protein UL69 are known to contain human T-helper epitopes (Beninga et al., 1995).
One approach to the development of a subunit HCMV vaccine is the use of live viral vectors to express relevant HCMV gene products.
It can thus be appreciated that provision of a CMV or an HCMV recombinant poxvirus, and of compositions and products therefrom particularly NYVAC or ALVAC based CMV or HCMV recombinants and compositions and products therefrom, especially such recombinants containing coding for any or all of HCMVgB, gH, gL, pp150, pp65 and IE1, including recombinants expressing altered or truncated versions of IE1 and/or gB and compositions and products therefrom would be a highly desirable advance over the current state of technology.